Bhavsar J H, Remmler J, Lobel P
Rutgers University, New Brunswick, New Jersey 08903.
Anal Biochem. 1994 Sep;221(2):234-42. doi: 10.1006/abio.1994.1407.
We find that in protein blotting, the electrophoretic transfer efficiency of a given protein can be markedly affected by other components in the sample. Using a variety of electrophoretic elution protocols, we obtained very inefficient transfer of purified 125I-labeled 275-kDa cation-independent mannose 6-phosphate receptor from a sodium dodecyl sulfate-polyacrylamide gel to a membrane. Surprisingly, the transfer efficiency increased significantly if other proteins that migrated in the vicinity of the receptor were present. This may be a generally unappreciated but potentially widespread phenomenon as other but not all radiolabeled proteins examined behaved similarly. This carrier effect occurs under conditions typically used in protein blotting experiments and can lead to large errors in quantitative analysis. We developed two methods to minimize the carrier effect. First, addition of excess carrier protein to samples prior to electrophoresis saturated the carrier effect so that different samples had similar transfer efficiencies. Second, addition of either bovine serum albumin or sodium deoxycholate to the cathodic electro-elution buffer markedly increased transfer efficiency of radiolabeled proteins that exhibited poor transfer efficiency and minimized transfer differences between samples that contained or lacked carrier. These methods should be generally applicable to standard protein blotting and analysis protocols.
我们发现，在蛋白质印迹法中，给定蛋白质的电泳转移效率会受到样品中其他成分的显著影响。使用多种电泳洗脱方案，我们从十二烷基硫酸钠-聚丙烯酰胺凝胶向膜转移纯化的125I标记的275-kDa不依赖阳离子的甘露糖6-磷酸受体时，转移效率非常低。令人惊讶的是，如果受体附近迁移的其他蛋白质存在，转移效率会显著提高。这可能是一个普遍未被认识到但潜在广泛存在的现象，因为所检测的其他但并非所有放射性标记蛋白质都表现出类似行为。这种载体效应在蛋白质印迹实验通常使用的条件下会发生，并可能导致定量分析中出现较大误差。我们开发了两种方法来最小化载体效应。首先，在电泳前向样品中添加过量载体蛋白可使载体效应饱和，从而使不同样品具有相似的转移效率。其次，向阴极电洗脱缓冲液中添加牛血清白蛋白或脱氧胆酸钠可显著提高转移效率差的放射性标记蛋白质的转移效率，并最小化含或不含载体的样品之间的转移差异。这些方法通常应适用于标准蛋白质印迹和分析方案。
